Parasite-derived resistance

ABSTRACT

A method for conferring resistance to a parasite to a host of the parasite, which comprises isolating a gene fragment from the parasite and inserting the gene fragment or a DNA or RNA segment substantially homologous to the gene fragment or to a DNA or RNA sequence functionally equivalent to the gene fragment into the host, wherein (1) transcription of the gene fragment or the DNA or RNA segment in the host occurs in an anti-sense direction, (2) the gene fragment or the DNA or RNA segment is expressed as a gene product in the host, wherein the gene product is capable of disrupting an essential activity of the parasite, or (3) the gene fragment or the DNA or RNA segment is a binding site capable of competing with a native binding site in the parasite, is disclosed along with hosts produced by this process. Particularly preferred is conferring resistance using a gene fragment from a replicase gene of an RNA virus.

This application is a continuation of application Ser. No. 08/068,168,filed on May 28, 1993, now abandoned, which is a continuation ofapplication Ser. No. 07/856,889, filed on Mar. 25, 1992 (now U.S. Pat.No. 5,240,841), which is a continuation of application Ser. No.07/449,049, filed on Dec. 14, 1989, now abandoned, which is acontinuation of application Ser. No. 06/842,484, filed on Mar. 21, 1986,now abandoned, which is a continuation-in-part of application Ser. No.06/714,263, filed on Mar. 21, 1985, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods of conferring resistance toparasites, such as viruses, bacteria, and higher parasites, to hosts ofthe parasite. More particularly, the present invention relates to vitalresistance obtained by genetic engineering of the host organism tocontain a portion of a replicase enzyme from an RNA virus.

2. Description of the Background

A potentially important application of genetic engineering technology isin the area of producing resistance to parasites. The proposals in theprior art that have been systematic and broadly applicable have centeredon finding a gene conferring resistance within a strain of the hostspecies or within a related species and transforming the gene into thegenome of a susceptible host. This approach may prove effective but hasseveral distinct disadvantages. Resistant forms of the host may notexist or may be very difficult to find for each new race of parasitewhich arises. Such resistance may be polygenic, making the cloning andtransfer of the resistance genes difficult. Where resistance is encodedby a gene, there are commonly already strains of the parasite that haveevolved virulence genes for overcoming such host-derived resistances ina gene-for-gene fashion (Flor 1971). Finally, the problem of identifyingand isolating the resistance gene from within the large genome of thehost will generally remain very difficult. An alternative strategy thataddresses these problems is therefore needed.

There have also been proposals for and some work on using genes fromorganisms unrelated to either host or parasite, which serendipitouslyhave gene products detrimental to a specific parasite. The gene codingfor the endotoxin of Baccillus thuringliensis (which is toxic tolepidopterous insects) would be an example of this (Held et al., 1982).While this type of approach may prove useful in some specific cases, itclearly represents an opportunistic approach to the problem, as opposedto a systematic methodology that can be applied very broadly.

There already exist some examples of .genes, gene derivatives, or geneproducts of a parasite that can produce a negative interaction withitself or a related genotype. Studies into the susceptibility of plantsto infection by viruses have demonstrated that closely related plantviruses or different strains of the same virus will cross-protect a hostorganism (Hamilton, 1980). In other words, a plant infected by a firstvirus is often not subject to infection by a second strain of that virusor by a related virus. A similar phenomenon has been observed in animalviruses and has been termed intrinsic interference (Marcus and Carrier,1967). From the point of view of parasite resistance of the typediscussed herein, the key proteins involved in the intrinsicinterference phenomenon are the viral replicase proteins (Marcus andZuckerbraun, 1970). These same authors proposed that the replicaseproteins of the primary infecting virus prevent the replication of thesecond virus by binding to its replicase attachment sites (Marcus andZuckerbraun, 1969). A similar proposal has been put forth to explaincross-protection in plants (Gibbs, 1969). In a similar manner,experimenters working with an E. coli infected with bacteriaphage 434have found that infected bacteria are immune to other phages (Lauer etal, 1981; Flashman, 1978; Roberts et al, 1979). Other workers havenoticed that endogenous as well as experimentally introducedcomplementary oligonucleotides can interact with mRNA in a potentiallydetrimental manner. Simons and coworkers (1983) have suggested thathybridization of a small anti-sense transcript to E. coli Tn10 mRNAcontributes to the regulation of transposition of that element.Stephenson and Zamecnik (1978) and Zamecnik and Stephenson (1978) haveshown that synthetic oligodeoxynucleotides, complementary to Roussarcoma virus terminal repeats, diminish normal viral infection and caninhibit viral RNA translation in vitro. However, these discoveries werenot applied to the production of host resistance to a parasite.

Despite this fragmentary knowledge in the prior art, there still remainsa need for a fully developed technique for producing resistance toparasites that is not based on the traditional methods of using aresistance gene from an immune strain of a host.

SUMMARY OF THE INVENTION

According, it is an object of this invention to provide a method ofconferring resistance to a parasite (specifically an RNA virus) to ahost of the parasite which does not rely on the necessity of identifyingand isolating a resistance gene from an immune strain of the host.

This and other objects of the invention as will hereinafter become morereadily apparent have been accomplished by providing a method forconferring resistance to a parasite to a host of said parasite, whichcomprises isolating a gene fragment from an RNA virus, wherein the genefrom which said gene fragment is derived codes for a replicase enzyme,and inserting said gene fragment or a DNA segment substantiallyhomologous to at least a part of said gene fragment into said host,wherein said gene fragment or DNA segment is expressed as a peptide insaid host, wherein said peptide is capable of binding to a replicasebinding site in said host.

BRIEF DESCRIPTION OF THE DRAWING

The FIGURE shows in schematic form the replicase gene from Qβ and itscleavage sites as described in detail in this application as well as thelocation of the replicase insert in plasmid pUC18.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The concept off parasite-derived resistance is that host resistance to aparticular parasite can effectively be engineered by introducing a gene,gene fragment, or modified gene or gene fragment of the pathogen intothe host. This approach is based upon the fact that in any parasite-hostinteraction, there are certain parasite-encoded cellular functions(activities) that are essential to the parasite but not to the host. Anessential function is one which must operate if the parasite is tosurvive or reproduce. These functions represent the Achilles heel of theparasite. If one of these functions is disrupted, the parasitic processwill be stopped. "Disruption" refers to any change that diminishes thesurvival, reproduction, or infectivity of the parasite. Such essentialfunctions, which are under the control of the parasite's genes, can bedisrupted by the presence of a corresponding gene product in the hostwhich is (1) dysfunctional, (2) in excess, or (3) appears in the wrongcontext or at the wrong developmental stage in the parasite's lifecycle. If such faulty signals are designed specifically for parasiticcell functions, they will have little effect on the host. Therefore,resistance to a particular pathogen can be achieved by cloning theappropriate parasite gene, if necessary modifying its expression, andtransforming it into the host genome. By resistance is meant anyreduction in virulence of the parasitic infection or any reduction inthe susceptibility of the host to the parasite.

This approach to engineering resistance has important advantages:

1) The source of resistance genes would never be in question, since eachparasite would bring with it the genes necessary for derivingresistance.

2) The stability of parasite-derived resistance will generally begreater than the stability of simply inherited forms of host resistance,for reasons that are discussed later in more detail.

3) The difficulties involved in cloning genes from host organisms, whichgenerally have larger genomes relative to their pathogens, are lessened.

4) Parasite-derived resistance will have a minimal effect on the hostand should not produce substances harmful to man.

The general concept of parasite-derived resistance is described in U.S.patent application Ser. No. 714,263, filed Mar. 21, 1985, which isherein incorporated by reference. The inventors have now specificallyreduced to practice a particular embodiment of the invention describedgenerally in the parent application. As will be described in detaillater, the specific embodiment is related to the replicase gene of anRNA virus.

All positive-strand RNA viruses that have been investigated contain anenzyme known as RNA replicase, which can utilize vital RNA as thetemplate for the formation of new RNA. RNA replicase is not normallypresent in a host cell, but is produced when the cell is infected withan RNA virus.

Vital RNA codes for formation of an RNA replicase which functions onlywith viral RNA as a template and ignores all other RNA molecules.Accordingly, the RNA replicase represents a vital function that is notmart of a normal host function and thereby represents a preferred meansof conferring resistance to a host using genes derived from the parasite(vital) organism.

Since replicase enzymes in both plant and animal RNA viruses havestriking sequence and functional similarities, the present inventionallows the production of resistance to a wide variety of RNA viruses ina simple and straight-forward manner. It is possible to use either thespecific replicase domain (peptide resulting from a gene fragment asdescribed herein) to confer resistance to other RNA viruses, or equallypossible to select from other viruses domains having functional homologyfor the domain of the Qβ virus used in the current reduction topractice.

The present invention is practiced by isolating a gene fragment from thereplicase gene of an RNA virus, preferably Qβ virus, and inserting thegene fragment or a DNA or RNA segment substantially homologous to atleast a part of the gene fragment or to a DNA or RNA sequencesubstantially equivalent to the gene fragment into the host, whereby thegene fragment or DNA or RNA segment is expressed as a gene product(peptide) in the host. In particular, a gene fragment is selected whichproduces a peptide containing the binding region (for RNA) of thereplicase enzyme. When the gene product, which is less than the entirereplicase enzyme and therefore is not capable of functioning as areplicase enzyme, binds to the RNA, it prevents binding of an activereplicase enzyme and therefore protects the host against the results ofinfection by an active virus.

The present reduction to practice was obtained using a gene fragmentfrom the Qβ virus and is therefore a preferred embodiment of theinvention. However, gene fragments from the replicase gene of other RNAviruses are equally usable in the method of the present invention.

When a Qβ replicase gene fragment is used, it is preferred to obtainthis fragment by cleaving Qβ replicase cDNA with Sau3a at the 5'terminal of the fragment and with NarI at the 3' terminal of thefragment. Use of these designated restriction enzymes produces fragmentsof the replicase gene of two types, approximately 500 and approximately1,000 base pairs in length. There are a number of spaced Sau3a cleavagesites in the replicase gene closely spaced from one another so thatthere are at least five fragments of approximately 1000 base pairs inlength and at least three fragments of approximately 500 base pairs inlength, all terminating at a NarI cleavage site. The NarI site is beyondthe transcriptional termination site of the replicase gene.

It is preferred to prepare a DNA vector by annealing a 5' Sau3a site inthe replicase coding region to a BamHI site in the lacZ gene on theplasmid pUC18. This creates a gene, when in-frame, encoding a fewamino-terminal amino acids of lacZ followed by a replicase proteindomain of varying sizes (depending on the specific Sau3a cleavage sitesselected) which terminates with a NarI site beyond the transcriptionaltermination site of the replicase gene. The production of the replicasedomain would then be under the lacZ promoter control. Some of the Sau3asites will produce an out-of-frame fusion of replicase, but suchinoperable gene products are outside the scope of the present inventionand can be readily detected since transformation with a vectorcontaining such gene products would not protect the host againstinfection with an RNA virus.

In addition to the specific vector described above, it is possible touse gene fragments and DNA segments coding for a peptide capable ofduplicating the binding function of the replicase enzyme using standardtechniques of genetic engineering and other vectors.

In addition to gene fragments obtained from natural sources of RNAreplicase enzymes, it is also possible to practice the method of thepresent invention using a DNA segment encoding a peptide substantiallyhomologous to a peptide encoded by the transcribed portion of the 3' endof an RNA replicase gene. Substantial homology preferably means at least90% of the amino acids in a given segment are identical (and preferablythat any substitutions are conservative substitutions), more preferablythat no more than two (preferably conservative) amino acids within agiven segment are different, and most preferably no more than one(preferably conservative) amino acid within a given segment isdifferent.

It is preferred that a DNA segment coding for such a homologous peptideor for a segment of a natural replicase enzyme contains from 400-1200base pairs (measured by the 3' end of the transcribed portion of an RNAreplicase gene), preferably from 500-1000 base pairs in length.

As an example of how disease resistance in general can be engineered bythe general approach, the discussion below sets forth in detail how thegenes of the bacteriophage Qβ can be used to make E. coli resistant toQβ infection. This example is not to be considered limiting of theinvention but is an example of the ease with which the invention can bepracticed, now that it is disclosed.

The biology of Qβ and other RNA phages has been extensively documented(Zinder, 1975), and the cDNA sequence of its genome has been determined.The Qβ genome has three major cistrons. These code for a maturationprotein (involved in lysis and phage binding to host pili), a coatprotein, and a subunit of the replicase enzyme. (A fourth gene productis a minor coat protein which is a read-through product of the coatcistron.

The life cycle of Qβ is basically as follows. The phage begins bybinding to the sex pili of F' E. coli, through which it enters the celland begins to translate its replicase subunit. Its replicase subunitpolymerizes with three host subunits normally involved in host proteintranslation. The resulting hybrid tetrameric enzyme has RNA replicaseactivity specific for Qβ. This specificity is due to the affinitybetween the Qβ subunit of the tetrameric replicase and a short segmentof the Qβ genome within the replicase cistron, The replicase attaches toQβ RNA at this binding site and replicates the viral RNA. Late in thelife cycle of Qβ, coat protein and maturation protein accumulate in thehost. The coat protein then binds to the replicase cistron and therebyrepresses translation of the replicase subunit. Termination ofreplication allows viral assembly, and eventually the maturation proteinlyses the host, releasing a new population of infective Qβ.

From a conventional (prior art) perspective, the life cycle of Qβsuggests two potential mechanisms for developing resistance.Host-derived resistance might be developed by (1) blocking Qβ binding tosex pili or (2) producing variant host subunits lacking affinity for theQβ replicase subunit. Blocking Qβ binding is, in fact, a known mechanismfor producing Qβ resistance, since non-F' routants lacking pili areimmune to infection (Silverman, Rosenthal, Mobach and Valentine, 1968)However, this strategy clearly disrupts a mechanism which is relevant tothe host's fitness as a species. The selection of variant forms of thehost subunits which help make up the replicase enzyme may also be anaturally occurring mechanism conferring resistance. Since the hostsupplies 3 of the 4 subunits of the viral replicase, one might expectmutations within these genes to confer resistance. However, the extentto which these host subunits can be altered is clearly limited, sincethese subunits are essential to host protein synthesis and the survivalof the host. Most of the variants of these host subunits would probablybe lethal or sub-lethal for the host. Even non-lethal variants arelikely to be suboptimal for protein translation efficiency. Therefore,both of the host-derived resistance mechanisms suggested by the Qβ lifecycle would be obtained at the expense of disrupting crucial hostfunctions.

The prospect of being able to transfer genes from parasite to hostprovides a new approach to resistance. Viewed from this perspective, thelife cycle of Qβ suggests at least as many mechanisms ofpathogen-derived resistance as host-derived resistance. Severalstrategies are seen to be promising: (1) deriving resistance from the Qβcoat protein; (2) deriving resistance from a modified Qβ replicase; (3)deriving resistance by cloning the Qβ replicase binding site, and (4)deriving resistance from expression of anti-sense strand RNA sequences.Another strategy involving the maturation protein also appears feasible.

Resistance Derived from the Coat Protein

Qβ coat protein is known to have a regulatory, as well as a structuralrole. Late in the phage life cycle, coat protein binds to and repressesthe cistron coding for the Qβ replicase subunit, stopping replicationand allowing vital assembly (Bernardi and Spahr, 1972). When cDNA to thecoat protein translational sequence is linked to an E. coli promoter andintroduced into E. coli, the coat protein is produced in the host.Expression of coat protein (in sufficient quantity) in the host willrepress replication of any infecting Qβ, thereby conferring resistanceon the transformed host.

Resistance Derived from a Derivative of the Replicase Gene

The Qβ replicase subunit has a dual affinity for a segment of the Qβgenome and the three host replicase subunits (Kamen, 1970; Meyer,Webster and Weissmann, 1981). If the Qβ replicase gene is cloned (ascDNA) and mutagenized, some variant forms will be able to bind to the Qβreplicase site and at the same time fail to polymerize with the hostsubunits, a requirement to form a functional replicase. Alternatively, aportion of the replicase gene can be cloned to produce a polypeptidecontaining the functional domain for binding the replicase site butincapable of interacting with the host subunits. A transformed hostproducing such a modified replicase subunit would be Qβ-resistant if themodified Qβ replicase subunit or a portion of it binds to thereplication sites of infecting Qβ and effectively competes with nativeQβ replicase for binding sites, thus disrupting Qβ replication. Detailsof this strategy are set forth in other locations of this specification.

Resistance Derived from Cloned Replicase Binding-Site

The above-mentioned replicase binds to a specific segment of the Qβgenome which is roughly 100 base pairs in length. If this segment iscloned (cDNA) and introduced into the host, it would be transcribedconstitutively as mRNA if attached to an appropriate promoter. Thetransformed host would then be resistant to Qβ because the binding site,which has been shown to compete for binding of the replicase enzyme invitro (Meyer, Weber and Weissmann, 1976), would limit the free replicaseavailable for Qβ replication.

Anti-sense Strand Interference

The presence of an RNA complementary to Qβ RNA would allow formation ofan RNA--RNA duplex that would block Qβ infection. This can beaccomplished, for example, by transcribing a cDNA clone of a portion ofQβ in the reverse orientation in the E. coli host. The anti-sense strandRNA produced will then hybridize to the infecting Qβ and interlet withits proper translation or packaging. The advantages of this approach arethat potentially any fragment of the vital genome could be used withoutmodification, and it would be extremely difficult for the virus toovercome this form of resistance.

Resistance Derived from Qβ Maturation Protein

Although the maturation protein's mode of action is not yet wellunderstood (Karik and Billeter, 1983; Winter and Gold, 1983), it alsorepresents a potential source of pathogen-derived resistance. A modifiedmaturation protein in the host can block lysis. Alternatively, arepressed operon containing a wild-type maturation gene can beengineered in the host that would be activated by Qβ infection. Thiswould induce premature lysis of a host cell upon initial infection byQβ, constituting (on the population level) a form of hypersensitivity.

Although the examples set forth above describing methods by whichbacteria can be protected from bacteria phage Qβ are related inparticular to a specific host/parasite system, the techniques arereadily applicable to other systems, such as the protection of otherorganisms from both viral and non-viral infections. Techniques forachieving these results are set forth in more detail in the followingparagraphs.

Virus Resistance

The most likely early application of the concept of parasite-derivedresistance is in engineering virus resistance. This is because the vitalgenome is small, and, since virus only propagates in the host, most ofthe genome is involved in pathenogenicity. Portions of the vital genomecan be cloned and their potential for conferring resistance readilydetermined. Alternatively, resistance-conferring genes can be discoveredempirically by testing the biological effect of various DNA restrictionfragments of the vital genome. Most virus-derived resistances are likelyto involve a block in replication. The methods described for engineeringresistance to Qβ are directly applicable to any virus which a) codes fora protein which helps regulate the virus' reproduction; b) has specificbinding sites in its genome; c) synthesizes its own replicase or reversetranscriptase; or d) is bound by complementary reverse strand sequencesof nucleic acid. In other words, these methods would apply toessentially all viruses.

While there has been some controversy among biochemists regardingwhether plant viruses encode their own replicase, it now seems likelythat most plant viruses do code for all or part of their replicases(Hall, Miller and Bujarski, 1982; Dorssers, Van der Meer, Kamen, Zabel,1983). The first plant virus to have its replication mechanismcharacterized, turnip yellows mosaic virus, has proven analogous to Qβ(Mouches, Candresse and Bove, 1984). This virus has been shown to have ahybrid replicase, with its own sub-unit conferring specific binding toits genome. This indicates that the approach described for Qβ replicasewould also apply to this virus. It is likely that most or all RNA plantviruses will code either for their own replicase, a subunit of thereplicase, or a protein modifying the specificity of the host's RNApolymerase. It is known that there is substantial homology betweenreplicases from a wide variety of RNA variety of RNA viruses (Kamen andArgos, 1984). This means that the replicase-derived resistance strategyoutlined for Qβ will be directly applicable to a wide range of plantviruses. Many viruses have not yet been analyzed relative to thisgenetic structure. However, the very small size of the vital genome andthe diversity of potential resistance mechanisms clearly indicates thata vital-derived resistance gene can be derived from any virus simply byusing standard shotgun cloning methods and direct screening forsubsequent resistance to the virus.

Non-vital Resistance

The application of parasite-derived resistance to extracellularparasites is more complex than for vital parasites. Since false signalscoded for by the host must be recognized by the parasite,parasite-derived resistance will only be useful where mechanisms existwhich allow recognition or incorporation by the parasite of non-degradedmacromolecules from the host. Van der Plank (1978) has offeredpersuasive theoretical arguments indicating that such an exchange ofmacromolecules between the host and the parasite often occurs. There isat least one case where such incorporation has been documented. In themalaria host/parasite system the parasite has been shown to incorporateand utilize a host dismutase enzyme, indicating the presence of aprotein exchange mechanism (Fairfield, Meshnick and Eaton, 1983). To theextent that such mechanisms exist in other non-viral host/parasiterelationships, the techniques described herein can be applied withoutsignificant modification. The existence of protein exchange mechanismscan be determined using monoclonal antibody probes to locatesub-cellular components, in conjunction with 2-D electrophoretic studiessearching for host-parasite hybrid proteins.

Given a macromolecular exchange mechanism, a variety of approaches tothe engineering of parasite-derived resistance exist for either vital ornon-vital parasites. For example, in gene-for-gene host/parasite systems(Flor, 1971; common in vital, fungal, and bacterial pathogens), it isgenerally found that the parasite's avirulence alleles are dominant tovirulence alleles (reviewed in Van der Plank, 1978). This suggests thatthe avirulence gene products override or block the activity of thevirulence gene products--thereby preventing infection. Thus, anavirulence allele cloned from an avirulent strain of the parasite, whenintroduced and expressed constitutively in a transformed host, can enterthe parasite or act at the host-parasite interface and override theinfective capacity of an otherwise virulent pathogen. Avirulence allelescan be identified by a variety of methods. For example, in bacteria thevirulence-avirulence locus can be cloned by using insertional mutation(employing transposable elements) of the virulent strain and screeningfor non-virulent mutants or by screening a genomic library forcomplementation of the virulence allele. The virulence gene can then beintroduced into the host to confer resistance. Recently, an avirulencegene has been cloned from the bacterial pathogen Pseudomonas syringae.However, the expressed intent of these workers is to clone theresistance gene from the host and the parasite gene has not beenintroduced into the host in any form (Staskawicz et al 1984). Thetechnique proposed here introduces an entirely new dimension to theclassical model of gene-for-gene host/parasite interactions.

Resistance from the Parasite's Regulatory Genes

A more general strategy for engineering parasite-derived resistance(applicable with or without gene-for-gene interactions) utilizesspecific regulatory genes from the parasite. For example, fungal genesregulating haustorial development or sporulation can be introduced intoa host, thereby disrupting the normal life cycle of the fungal pathogen,using established techniques of identifying the regulatory protein andsearching a genomic library with an antibody probe. Once cloned, suchgenes can be introduced into a host, where they will disrupt the normallife cycle of the fungal pathogen. This type of regulatory approachappears particularly useful in the engineering of insect resistance. Forexample, all insects depend on the regulated biosynthesis of juvenileand molting hormones for precise timing of molting, metamorphosis andreproduction. Using the techniques of this invention, it is possible toincorporate into the host genes from the insect pest encoding theactivities necessary to produce the insects' hormones, pheromones orneurotransmitters. In the case of neurotransmitters, these polypeptidesare typically extremely short (less than 20 amino acids) and aretherefore easily sequenced, and artificial genes coding for thesesequences can then be synthesized de novo. In the case of non-peptidehormones or pheromones the problem is more difficult but can beovercome. Typically, several enzymatic steps will be required from thestarting point of a common precursor in both host and parasite to thebiologically active secondary metabolite. This means that several genesin the parasite will have to be identified, cloned and transferred tothe host. While this approach does not have the simplicity or directnessof other parasite-derived approaches, it is potentially one of the moresignificant and broad-spectrum applications of parasite-derivedresistance, and will generally warrant the time and expense ofengineering the latter part of a biosynthetic pathway. The hostproducing such insect growth regulators or transmitters would beresistant by virtue of disrupting the behavior or life cycle of theinsect pathogen, thereby eliminating infection of the primary host.There are examples in nature where plants seemed to have exploited asimilar strategy for resistance by evolving genes producing analogs to,or biosynthetic antagonists of, insect hormones (Bowers, 1980).

Another application of parasite-derived resistance is available where aninsect or other organism serves as an intermediate host, so that thedisease cycle can be disrupted by making the intermediate host resistantto the pathogen, thereby eliminating infection of the primary host. Forexample, efforts to control malaria have previously focused oneradication of the intermediate host, the Anopheles mosquito. Ifhowever, genes from the Plasmodium pathogen are introduced into themosquito in a manner to confer resistance by disrupting the life cycleof the parasite, the disease cycle will be broken. This approach is mostfeasible if the resistance genes is of selective advantage to theintermediate host, allowing resistance genes to be maintained andpropagated in natural populations after introduction of modifiedindividuals. This can be done, for example, by concurrently introducingresistance to a pesticide into the intermediate host.

Advantages and Limits of Pathogen-derived Resistance

Parasite-derived resistance represents a systematic and broadly-relevantapproach to the problem of how to genetically engineer insect anddisease resistance. The rich possibilities of this approach areillustrated by the fact that three different strategies for derivingresistance from the Qβ bacteriophage exist in a parasite having onlythree genes. There are several distinct advantages of parasite-derivedresistance.

One of the most attractive features of parasite-derived resistance isthat each new parasite or race of parasite that becomes a problemsimultaneously brings with it the specific genes needed to engineerresistance to itself. These genes can be systematically identifiedwithin the parasite's genome. Once such genes have been identified,homologous genes in other parasite races or in related parasites will bereadily identifiable by DNA hybridization techniques. This eliminatesthe need for repeated and exhaustive searches through the host'sgermplasm pools, seeking rare host resistance genes.

Another major advantage of this strategy is that it should not generallybe disruptive of host functions. Van der Plank (1978), usingevolutionary arguments and population genetics data, has argued thathost genes controlling susceptibility exist because they involveessential host functions. Most hosts are genetically susceptible becausethe susceptible allele is optimal relative to its natural function.Host-derived resistance alleles, therefore, tend to disrupt the optimalfunctioning of the host. To the extent that this is true, mosthost-derived resistances attack the pathogen indirectly by replacing anoptimal host gene product with a non-optimal host gene product whichhappens to be incompatible with the parasite. This is seen in the Qβsystem, where host-derived resistance is likely to be achieved either bydisrupting sex pili formation or by tampering with the host'sprotein-synthesis machinery. A similar situation exists with sickle-cellanemia, which is harmful to humans when expressed but which confersresistance to malaria in persons who have both a recessive sickle-cellgene and a normal hemoglobin gene. The beauty of the concept ofpathogen-derived resistance is that only pathogenic cell functions areattacked and are attacked directly, which will have minimal subsequenteffect on the host. The specificity of parasite-derived resistance isnot only desirable in terms of being non-disruptive to the host, butalso of being non-harmful to man. Resistance based upon production ofgeneral toxicants, such as the natural pesticides of many resistantplant taxa, have been shown to be potentially harmful to man wheningested (Ames, 1983). The specificity of parasite-derived resistanceshould preclude, to a large extent, any such harm to man.

There are reasons to believe that parasite-derived resistance should berelatively durable compared to host-derived resistance. The ability ofparasites to circumvent host-generated general toxicants is well known.Additionally, specific host-derived resistance genes are frequentlyovercome by matching gene-for-gene mutations to virulence in theparasite (Flor, 1971). In the case of host-derived Qβ resistance,alterations in the host replicase sub-units (making them incompatiblewith the viral subunit, thereby conferring resistance), are easilymatched by mutations in the Qβ replicase subunit which restore subunitcompatability, constituting a mutation to virulence. However, suchgene-for-gene mutations circumventing resistance should be relativelyrare in the case of parasite-derived resistance. In this case theparasite would usually be facing a new form of resistance, which it hadnot previously faced in its evolution. These types of resistances arelikely to be very difficult for the parasite to overcome, especiallywhere regulatory genes are involved. For example, resistance to Qβ wasderived from the Qβ coat protein gene, a new virulent Qβ strain couldonly arise by first having a new binding site develop by mutation in thereplicase cistron (without disrupting replicase function) which wouldnot bind the native coat protein. Simultaneously a new coat proteinwould have to arise by mutation (without disrupting coat proteinfunction) which would bind to the new binding site. Such a simultaneousand complementary set of mutations (which preserved both coat andreplicase functions) should be extremely rare.

Last, engineering parasite-derived resistance should be considerablymore approachable on the molecular level than engineering host-derivedresistance. There are numerous reasons for this: (1) this strategy wouldgenerally focus on the molecular biology of relatively simple organismswith short life cycles; (2) it would generally require only theidentification and isolation of individual genes from small genomes; (3)unregulated, constitutive expression of the parasite-derived resistancegenes would usually be effective; and (4) it would avoid the complex,multigenic biosynthetic pathways which are the likely basis of manyexisting host-derived resistances.

There do not seem to be any obvious disadvantages to theparasite-derived approach to resistance, except that application of thestrategy to non-virus parasites is only possible where mechanisms existfor macromolecular exchange between host and parasite. Most forms ofparasitism, especially those forms displaying gene-for-gene resistance,allow ample opportunity for gene-product interactions and will besuitable for engineering parasite-derived resistance.

Techniques for the Production of Resistant Host

As will be readily understood that those of ordinary skill in the art ofgenetic engineering, standard techniques of genetic engineering canreadily be adopted to attain the goals set forth herein. Protection of ahost against a virus, for example, can easily be achieved. Because ofthe reasons set forth above, it is not necessary to identify the genebeing inserted into the host, although identification of the gene willmake application of the method easier to perform. In general, geneticinformation (DNA or RNA) from any virus is isolated using standardprocedures and cleaved into pieces of varying lengths, preferablycontaining at least 20 nucleotides if the DNA is to be transcribed in ananti-sense direction, or at least a functional portion and preferably anentire gene if the gene is to be expressed. DNA fragments are typicallyobtained using restriction endonuclease enzymes. The same enzyme (orenzymes) is then used to cleave a vector capable of replicating in thehost or inserting into a host's chromosome. The vector can be a naturalplasmid or transposon or any part thereof capable of replication in thehost and, when desired, production of a gene product from the exogenousparasite gene fragment. Vectors derived from plasmids and other vectorsnormally present in the host are preferred. The viral DNA is insertedinto the vector using standard techniques in either a sense direction(when expression of a gene product is desired) or an anti-sensedirection. Proper tailoring of the gene fragment in the vector (e.g.,employing appropriate 5' and 3' flanking sequences to ensure regulation,transcription, and translation as desired) is readily achieved usingstandard techniques, especially when simple constitutive expression isdesired, as is suitable in most cases of parasite-derived resistance. Asused in this application, the phrase "gene fragment" encompasses bothentire genes, DNA segments that contain an entire gene or a portionthereof, and segments of DNA that are incomplete parts of a single gene.The word "gene" encompasses both DNA sequences that code for a peptidegene product and other DNA sequences that form a functional part of achromosome or plasmid.

Although this specification generally refers to DNA alone whendescribing genetic information, vectors, or the like, this is done forease of expression only. Any reference to DNA, unless clearly restrictedto DNA and not to RNA, is equally applicable to RNA. For example,pathogenic RNA viruses can be the source of the parasite gene fragment,and non-virulent RNA viruses can act as vectors. In many instances,however, it is easier to work with DNA than RNA (e.g., more DNArestriction endonuclease enzymes are known), and use of cDNA preparedfrom RNA is a preferred embodiment of the invention when producingresistance to an RNA virus.

After a gene fragment has been isolated, the DNA sequence can bedetermined and modified, if desired, to produce similar DNA segmentscapable of being expressed as the same or similar gene products. Forexample, one or more codons can be replaced by equivalent codons toproduce artificial DNA segments coding for the identical gene product.Alternately, a codon can be replaced by a codon that codes for a similaramino acid (e.g., a codon for lucine replaced by a codon for isoleucineor a codon for glutamic acid replaced by a codon for aspartic acid).When used as an antisense strand or binding site, less than 10%non-identical nucleotides are preferred with unmodified gene fragmentsbeing most preferred. Greater modification of the gene fragment ispossible when a gene product of the parasite gene is being produced. Forexample, artificial DNA sequences containing a series of codonsfunctionally equivalent (i.e., that code for the same amino acids) tothe codon sequence in the parasite gene fragment are considered fullyequivalent to the parasite gene fragment since they will produce thesame gene product, even though the DNA sequence can be substantiallydifferent. Gene products not identical to the natural gene product butretaining the ability to produce a gene product capable of disrupting anessential activity of the parasite can be produced by systematicmodification of codons (and thus the expressed gene products) followedby testing for parasite resistance. Such modified DNA segments must besubstantially homologous to at least a part of the isolated genefragment or a DNA sequence functionally equivalent thereto in order tobe considered indicative of parasite-derived resistance. By "substantialhomology" is meant at least 80%, preferably at least 90%, and mostpreferably at least 95% identity between the DNA sequence in questionand the sequence to which it is being compared. Identical sequences arealso covered by the same phrase. Comparisons for the purpose ofdetermining homology are preferably made over a sequence of at least 15and more preferably at least 21 nucleotides.

The phrase "isolating a gene fragment", as used in this application,refers to the process of obtaining a gene fragment to be used in theproduction of resistance in a useful form. The gene fragment does nothave to be purified or otherwise separated from other cellularcomponents, although this will occur in many processes. Instead, theword "isolated" is used to indicate that a gene has been obtained in auseful form by a deliberate process. For example, an "isolated genefragment" can exist in a mixture of fragments from the DNA of a parasitethat is to be used in a shotgun cloning procedure. A gene fragment isalso still "isolated" when it is present in the form of a recombinantplasmid present in a bacterium being used in a shotgun cloning procedureto identify producers of desired parasite gene products (such as by useof monoclonal antibodies). Likewise, a segment of purified DNAcomprising a parasite gene segment termini from a cloning vector (e.g.,obtained by cloning a parasite gene fragment in a bacterial plasmidprior to insertion into the final host) is also encompassed by thisterm. Other usable forms of gene fragments will be readily apparent tothose skilled in genetic engineering.

Insertion of the parasite gene fragment into a host is readily achievedwhen the host is a bacterium or other unicellular organism since themajor advances that have occurred recently in genetic engineering havegenerally involved insertion of vectors containing exogenous genes intounicellular hosts (especially bacteria and yeasts) and are directlyapplicable to the present method. "Insertion" encompasses any means ofintroducing genetic information into a host organism compatible with thelimitations discussed in this specification. However, insertion in amanner to provide a heritable characteristic is preferred. In unicellarorganisms this can readily be accomplished using heritable plasmids orby insertion of the parasite gene fragment into the host chromosome.These examples are not limiting, and other methods of insertingheritable genetic information, whether into unicellar or higherorganisms, are equally applicable to the practice of this invention.

Proven methods for inserting new genes into higher organisms can now befound in a massive volume of current literature. There exist four basicmethods of doing this (Baserga, Crose, and Povera, Eds., 1980): (1)direct uptake of DNA or DNA-containing particles by the cell, (2) cellfusion with other cells or ghost cells, (3) microinjection, and (4)infective transformation. A fifth method is being developed whichinvolves the use of accelerated high-velocity one-micron-sized particlesfor the purpose of carrying DNA into cells and tissues.

Uptake mechanisms include the following: (1) induction of enhancedmembrane permeability by use of Ca⁺⁺ and temperature shock (Mandel andHiga, 1970; Dityakin et al., 1972); (2) use of surface binding agentssuch as PEG (Chang and Cohen, 1979; Krens et al., 1982) or Ca(PO₄)₂(Graham and van der Eb, 1973; Wiglet et al., 1979); and (3) phagocytosisof particles such as liposomes (Uchimaya et al., 1982), organelles(Potrykus, 1973), or bacteria (Cocking, 1972), into the cell. Theseuptake mechanisms generally involve suspensions of single cells, whereany existing cell wall materials have been removed enzymatically. Uptakeprotocols are generally quite simple and allow treatment of largenumbers of cells en masse. In such systems most cells are unaffected,but cell selection procedures are available to recover the rare cellsthat have been transformed (Powers and Cocking, 1977).

Fusion mechanisms incorporate new genetic material into a cell byallowing it to fuse with another cell. A variation on this themeinvolves ghost cells. The membrane of killed cells are allowed to fillwith a given DNA solution, such that cell fusion incorporates the DNAfrom the carrier "cell" into the living cell. Cell-to-cell fusion can beinduced with the aid of such things as PEG (Bajaj, 1982) and Sendaivirus particles (Uchida et al., 1980). As with uptake mechanisms, fusiontechnologies rely upon the use of single cell suspensions, where cellsare enzymatically stripped of any cell wall material. While fusiontechnologies can have relatively good efficiencies in terms of numbersof cells affected, the problems of cell selection can be more complex,and the resulting cells are typically of elevated ploidy.

Microinjection technologies employ extremely fine, drawn out capillarytubes, which are called microelectrodes. These can be made sufficientlysmall that they can be used as syringe needles for the direct injectionof biological substances into certain types of individual cells(Diacumakos, 1973; Graessmann and Graessmann, 1983). One modification ofmicroinjection involves pricking with a solid-glass drawn needle, whichcarries in biological solutions which are bathing the cell (Yamomoto etal., 1981). Another modification is called ionophoresis (Purres, 1981;Ocho et al, 1981), which uses electrophoresis of substances out of themicroelectrode and into the cell as an alternative to high pressure bulkflow. Microinjection procedures can give extremely high efficienciesrelative to delivery into the cell. Because of this, microinjection hasbeen used successfully in the transformation of individual egg cells.

In another example, foreign DNA was successfully injected into cottonpollen tubes without the pollen being damaged or its germination beinginhibited. Although this involved a resistance gene from another plantinstead of a parasite gene, the same technique can be used in thepractice of the present invention. DNA was injected into the nucleus ofcotton pollen grains germinating on cellophane using micro-manipulatorsand a micro-injection system. This operation was carried out on thefixed stage of an inverted research microscope equipped with Nomarskidifferential interference optics. Foreign DNA in a recipient nucleus wasdetected by epifluorescence after the incorporation of a fluorescentmarker in the injected material. The DNA was introduced using"quickfill" tubing drawn to a tip diameter of 0.5 micron, and the DNAwas injected into the nucleus iontophoretically. The germinating pollenwas returned to the style where it continued to grow and fertilize theovule. About 2N injections can be carried out per day. Seeds from themicro-injected plants were planted, and seedlings were raised andscreened. Screening may be carried out by testing for the presence ofthe foreign gene by Southern blotting or for the presence of the geneproduct by means of enzyme inhibition assays. In addition, screening forinsect resistance of the developing square and boll can be utilized whencotton is the host. Other plants can be treated in the same manner.

Infective transformation employs non-injurious infective agents of thehost, such as viruses, which naturally transmit part of their genomeinto the host. In plants, the principal mode of transformation now beingpracticed is the use of the infective agent Agrobacterium tumefaciens.This bacterium will naturally colonize cells of any dicotyledonous plantand transmit a specific "T-region" of its Ti-plasmid into the plantchromosome. Other plant vectors useful for the transformation of plantscan similarly be used. Genes of interest can now be routinely engineeredinto the T-region and can be transmitted to the plant by the bacterium(see Fraley et al., 1983). Simple conincubation (growing plant cells andbacterial cells together) has been shown to be extremely effective intransforming plant protoplasts and leaf disks, and whole transformedplants have now been regenerated in numerous plant species (see Horschet al., 1984). In mammals, naturally infective retroviruses have beenused to construct naturally transforming vectors which insert engineeredDNA into the mammalian chromosome, in a manner similar to Agrobacteriumtumefaciens. This transformation mechanism is considered extremelypromising for animal and human gene therapy (see Anderson, 1984).

For an example of mammalian transformation, see U.S. Pat. No. 4,396,601to Salser et al., which describes a technique in which cells areisolated from a regenerative body member of a mammal or a syngeneicequivalent to provide parent cells. The parent cells are combined withDNA from the parasite and with additional DNA that produces a aselection advantage over the parent cells when the cells are subjectedto mitotic inhibition. The modified cells are then introduced into thehost in a manner such that the modified cells return to the body memberfrom which the parent cells were obtained. A mitotic inhibitor is thenadministered to the host to provide a selective advantage for themodified cells over the parent cells, thereby regenerating the modifiedcells in the host. Further details of this method can be obtained byreference to U.S. Pat. No. 4,396,601.

The method of the invention is generally applicable to the protection ofany host from a parasite of that host. As used herein, "host" refers toany organism that can be infected by any parasitic or symbioticorganism. The term "parasite" refers to any organism that obtainssubstance or means for reproduction from an organism, whether it liveswith that organism in a parasitic or symbiotic relationship. Theparasite need not be specific for a particular host but may be aparasite of many hosts, such as the caterpillars of numerous moths andbufferflies. Although a preferable parasite for use in this invention isa virus, whether the virus is a DNA or RNA virus, other parasites arealso encompassed by this term. Examples of other parasites includebacteria, protozoa, fungi, nemotodes, insects, and arachnids.

Since a host is normally higher in the evolutionary scheme than theparasite, the term "host" does not encompass a virus, which resides atthe bottom of the evolutionary scheme. However, any higher organism iscapable of being infected by a parasite. The invention is readilyapplicable, for example, to bacteria grown in culture which needprotection against infection from bacteriophages. Additionally, plantsand other higher organisms, such as mammals, also can be readilyprotected from viruses using the method of the invention. Both plantsand animals can also be protected from higher parasitic hosts, such asinsects and protozoans, subject to the restrictions which have alreadybeen discussed. Examples of hosts include bacteria, yeasts, lungi (e.g.,mushrooms), legumious plants (e.g., soybeans), cereal and forage crops(e.g., corn, wheat, rice and alfalfa), food crops (e.g., tomatoes,potatoes, lettuce, and onions), ornamental plants (e.g., roses,junipers, and orchids), trees (e.g., pine, spruce, and walnut),protozoans amphibians, reptiles, birds (e.g., chickens and turkeys), andmammals (e.g., cats, dogs, horses, cattle, sheep, goats, pigs, andprimates).

Examples of host/parasite systems in which either the host or theparasite is a unicellular organism (the most common situations) can befound in numerous microbiology textbooks and reference manuals, such asCRC Handbook of Microbiology, Condensed Edition, Laskin and Lechevalier(eds.), CRC Press, Cleveland, Ohio, 1974. Other examples ofhost/parasite systems are given below along with examples of howresistance to the parasite can be given to the host in that system.These examples are not limiting, and many other methods for achievingresistance are possible for each listed system.

1) There are a variety of bacteria important in industrial fermentationprocesses, such as Streptococcus lactis, Streptococens cremoris, andLactobacillus species. During fermentation, infection by variousbacteriophages is a common cause of fermentation failure. Bacterialresistance to such bacteriophage infection can be engineered by methodsexactly analogous to the methods described above for engineeringresistance to the Qβ bacteriophage in E. coli.

2) There are hundreds of significant plant RNA viruses, and essentiallyall crop species are affected by one or more such viruses. Resistance tosuch viruses can be obtained in a manner closely analogous to Qβresistance in bacteria, by cloning fragments of the viruses intoplant-transforming vectors such as a modified Ti-plasmid andtransforming the appropriate plants. Plants transformed by various genefragments can then be screened for resistance, using established plantbreeding techniques. A few relevant viruses include alfalfa mosaicvirus, brome mosaic virus, barley yellow dwarf virus, beet yellowsvirus, cucumber mosaic virus, lettuce necrotic yellows virus, maizechlorotic dwarf virus, pea enation virus, potato viruses S, X, and Y,southern bean mosaic virus, tomato ringspot virus, tobacco ringspotvirus, tobacco mosaic virus, tobacco streak virus, turnip yellow mosaicvirus, and wound tumor virus.

3) There are certain animal and human pathogens, such as the flu andcommon cold viruses, which have evolved mechanisms for circumventing theeffectiveness of the animal immune system. Where such a virus is achronic problem, as with flu and colds, parasite-derived resistance willbe a powerful tool for conferring immunity to all strains of thatpathogen. Resistance can be engineered by cloning fragments of the viralgenome, introducing the gene fragments into animal cells in vitro by useof retrovital vectors, testing of varius tranformed cell times todetermine which have conferred resistance to infection by the virus, andthen using those fragments conferring resistance to create benignnon-infectious retrovirus vectors for the purpose of introducingresistance genes into individuals.

4) There are certain retroviruses which attack T-cells (i.e., the humanimmune system) directly (such as the viruses that produce AIDS), therebycircumventing our natural immune defense mechanism. Resistance can beengineered as described above, using AIDS genomic fragments, and alsousing AIDS, or a similar retrovirus, for the construction of aT-cell-specific transforming vector. Transformed T-cells withresistance-conferring fragments of the AIDS genome would have aselective advantage over other susceptible T-cells, becoming thepredominant form of T-cell and thereby giving rise to resistantindividuals.

5) A wide range of bacteria and fungi that parasitize plants haveintimate contact with living host cells and reveal gene-for-gene hostparasite relations. Resistance in such cases can be engineered bycloning avirulence alleles from avirulent strains of the parasite andintroducing these genes into the relevant host for the purpose ofconferring resistance. A few pathogens where this method is relevantinclude Puccinia sorghi infection of corn, Puccinia infections of wheat,Phytophthora infestans infection of potato, Ustilago infection of rye,and Melampsora lini infection of flax.

6) A wide range of insects parasitize plants, causing severe economiclosses, and depend upon a proper balance of juvenile hormone and moltinghormone to regulate their development. Therefore, broad-spectrum,insect-derived plant resistance to insects can be engineered by cloningthe insect genes responsible for the final steps of the biosynthesis ofthese hormones and transferring these genes to the plant hosts ofinterest, using established transformation techniques. Typical geneswould code for enzymes controlling the conversion of a precursor intothe desired regulatory product (e.g., hormone). Basically all planthosts contain the precursors for the synthesis of these hormones; i.e.,famesol in the case of juvenile hormone and phytosterols in the case ofmolting hormone. Other useful genes would be those producing otherregulatory substances that trigger the production of hormones inparasites. A few insect parasites which could be controlled by thismethod include flea beetles, wire worms, cutworms, grubs, aphids,leafhoppers, tarnished plant bugs, Colorado potato beetles, cucumberbeetles, weevils, cabbage worm, cabbage lopper, leafminers, Hessian fly,grasshopper, tent worm, gypsy moth, tussock moth, army worm, corn earworm, European corn borer, and Japanese beetle.

7) A wide range of insects parasitize plants and containneurotransmitters which control essential body functions. Suchneurotransmitters are oligopeptides typically only 5-20 amino acidslong. In this case insect-derived resistance can be engineered bysequencing the oligopeptide and synthesizing artificial genes homologousto the native insect genes coding for these neurotransmitters. Thesesynthetic genes, when expressed in a plant host, can then disrupt thatcrucial body function normally regulated by that neurotransmitter of theinsect parasite. The insect listed in the previous example would beequally valid as candidates for this method of deriving parasite-derivedresistance.

In addition to the above general procedures which can be used forpreparing recombinant DNA molecules and transformed unicellularorganisms in accordance with the practices of this invention, otherknown techniques and modifications thereof can be used in carrying outthe practice of the invention. In particular, techniques relating togenetic engineering have recently undergone explosive growth anddevelopment. Many recent U.S. patents disclose plasmids, geneticallyengineered microorganisms, and methods of conducting genetic engineeringwhich can be used in the practice of the present invention. For example,U.S. Pat. No. 4,273,875 discloses a plasmid and a process of isolatingthe same. U.S. Pat. No. 4,304,863 discloses a process for producingbacteria by genetic engineering in which a hybrid plasmid is constructedand used to transform a bacterial host. U.S. Pat. No. 4,419,450discloses a plasmid useful as a cloning vehicle in recombinant DNA work.U.S. Pat. No. 4,362,867 discloses recombinant cDNA construction methodsand hybrid nucleotides produced thereby which are useful in cloningprocesses. U.S. Pat. No. 4,403,036 discloses genetic reagents forgenerating plasmids containing multiple copies of DNA segments. U.S.Pat. No. 4,363,877 discloses recombinant DNA transfer vectors. U.S. Pat.No. 4,356,270 discloses a recombinant DNA cloning vehicle and is aparticularly useful disclosure for those with limited experience in thearea of genetic engineering since it defines many of the terms used ingenetic engineering and the basic processes used therein. U.S. Pat. No.4,336,336 discloses a fused gene and a method of making the same. U.S.Pat. No. 4,349,629 discloses plasmid vectors and the production and usethereof. U.S. Pat. No. 4,332,901 discloses a cloning vector useful inrecombinant DNA. Although some of these patents are directed to theproduction of a particular gene product that is not within the scope ofthe present invention, the procedures described therein can easily bemodified to the practice of the invention described in thisspecification by those skilled in the art of genetic engineering.

All of the patents and other publications cited in this specificationare indicative of the level of skill and knowledge of those skilled inthe arts to which the present invention pertains. All publications,whether patents or otherwise, referred to previously or later in thisspecification are herein separately incorporated by reference. Althoughfull incorporation of the individual publications is intended, it isrecognized that those of ordinary skill in the art can readily determinefrom the incorporated publications those sections which are mostrelevant to the present invention and those sections which could bedeleted without loss of understanding.

In addition to the method of producing resistance to a parasitedescribed above in detail, this invention also encompasses hostsproduced by the process of the invention as well as recombinant vectorsand other products of genetic engineering useful in the practice of theinvention.

The invention now being generally described, the same will be betterunderstood by reference to certain specific examples which are includedherein for purposes of illustration only and are not intended to belimiting of the invention or any embodiment thereof, unless sospecified.

EXAMPLE

The feasibility of the concept outlined above was proven withexperiments using the bacteriophage Qβ and its host, E. coli. Using cDNAclones of Qβ (Qβ is an RNA phage), plasmids were first constructed orobtained which would express part of the Qβ cDNA in E. coli and conferresistance.

Coat Protein: The plasmid used for production of the coat protein waspGL101 obtained from R. B. Winter (Cell 33, 877). This plasmid expressesthe coat protein under lac operator control, so its expression can beinduced by IPTG (though there is also a constitutive expression). Thisplasmid as well as the others described below contain the gene encodingamp^(r).

Negative Strand: A plasmid was constructed that inserted the 0.9 KbHpaII fragment of Qβ cDNA into pUR222 plasmid at the AccI site. Thisfragment extends from positions 2547 to 3473 in Qβ (FIGURE) and includestranslational sequences of the replicase gene. These sequences alsocontain the M-binding site of the replicase. In the "sense" orientationof this fragment, a fusion product between β-galactosidase protein andthe replicase fragment is formed. In the antisense (reverse) ligation ofthis fragment, an RNA complementary to the Qβ RNA sequence is formed.Both constructions were made.

Testing for Resistance: The strain GM1 (provided by R. W. Webster) wastransformed with pUR222 or one of the test plasmids described above.These strains were grown up, made competent, incubated with Qβ and thenplated out in soft agar. Plaque numbers and sizes were assessed todetermine if resistance was taking place.

In an initial experiment to test the coat protein, GM1+pUR227. andGN1+pGL101 were grown in 10 mls L-broth containing ampicillin in IPTG.At stationary phase the cultures were pelleted and resuspended in 4 ml50 mM YCaCl₂. A small portion, 0.1 ml, of this plating culture wasincubated 60' with 10⁷ pfu of Qβ. This was then plated on YT-ANP platesin 3 ml soft agar with IPIG. The results were that the GM1+pUR222 plateshad thousands of plaques which soon (24 hrs) engulfed the plate; theGN1+pGL101 plate at first showed no plaques but later developed manyvery small plaques.

To check the possibility that the GM1 strain+pGL101 resistance was dueto loss of the F' element, the strains were subsequently grown onminimal medium lacking proline to maintain selection for the F'. Thesame protocol as above was then repeated, including strains of GN1 withthe HpaII (sense) and HpaII (antisense) bearing plasmids. The resultsare presented in Table 1. Both the coat protein and the HpaII(antisense) plasmids could confer resistance to Qβ infection. Thisexperiment was repeated twice with essentially the same results. Aftercontinued passage, however, the plasmid bearing Qβ cDNA sequencesrearranged or were lost. Additionally, the pGL101 was tested at highertiter (10¹¹ pfu); it still conferred resistance. Coat-conferredresistance from the RNA phages f1 and f2 were tested. GN1 with pGL101was resistant to f2 but not f1 as might be expected considering theirmodes of infection.

                  TABLE 1                                                         ______________________________________                                        Strain + Plasmid                                                                           Inducer  # of Plaques                                                                             Size                                         ______________________________________                                        GM1  --          +        300      normal                                          puR222      -        360       "                                               "          +        348       "                                              Hpa (antisense)                                                                           -        247       "                                               "          +        263      small                                           Hpa (sense) -        224      normal                                           "          +        234       "                                              pGL101      -        176      very small                                       "          +        101      very very small                            ______________________________________                                    

Replicase Binding Domain

In an experiment similar to those described above, a model systeminvolving E. Coli and its viral pathogen Qβ was utilized. A pBR322plasmid containing a cDNA clone from the 3' end of the Qβ genome wasobtained from Martin Billeter at the University of Zurich. The genesegment of this plasmid encodes all of the replicase gene of Qβ. VariousDNA constructions were made using this source of a gene encoding the Qβviral replicase. The constructs were made by annealing a 5' Sau3a sitein the replicase coding region to a BamHI site in the lacZ gene on thepUC18 plasmid. The pUC18 plasmid is commercially available from BethesdaResearch Laboratories. The restriction enzymes used in producing thefragments as well as various other enzyme used in the geneticengineering steps described herein are also commercially available.

This process created a gene, when in-frame, encoding a fewamino-terminal amino acids of lacZ followed by a replicase proteindomain terminating with a NarI site beyond the transcriptionaltermination site of the replicase enzyme. Since the restriction enzymeSau3a recognizes a large number (approximately 8) of cleavage sites inthe replicase gene, a number of different DNA constructs were produced.The plasmids produced in this manner resulted in the production of thereplicase domain being under lacZ promoter control. Some of the Sau3asites in the replicase gene produced an out-of-frame fusion ofreplicase.

The vector constructions so made were transformed into E. Coli JM103, acommercially available strain. The size of the replicase gene fragmentin each construction was determined, and the susceptibility of eachtransformant to Qβ infection was tested.

Basically, there were two classes of fusions. One class containedapproximately 1,000 base pairs of the 3' end of the replicase (4 out of5 should be in-frame) and one class contained approximately 500 basepairs of the 3' end (2 of 3 would be out-of-frame). The 1,000-BP classwas represented by clones #7, #10 and 19011, while the 500-BP class wasrepresented by #4, #5, and #8 (see FIGURE). The colonies containingthese constructions were grown in L-broth and then in M-9 media toselect for male E. coli. Both of these media are commercially availableand are described in Maniatis et al, Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y.(1982). A plating culture was then made either with or withoutIPTG-induction (IPTG is isopropyl thiogalactoside). It was initiallysuspected that induction of lacZ expression with IPTG would be needed toproduce enough replicase protein fragment in order to see protectionfrom infection. However, as shown below, this did not prove to benecessary.

The results of the comparative tests are seen in Table 1 below. PlasmidpUC9 is JM103 with the parental plasmid which served as a negativecontrol. The reference 2290 is to the originalwhole-replicase-containing plasmid in JM103. All of the replicase fusionproducts produced a 10-fold or more protection against infection exceptconstruction #8. This construction had the smallest portion of thereplicase gene and may not be in-frame. All of the strains were aboutequally infectable as determined by infection with a differentpilus-specific phage, f2.

                  TABLE I                                                         ______________________________________                                                        No. of plaques/plate                                          Strain with Plasmid                                                                             -IPTG     +IPTG    f2                                       ______________________________________                                                PUC9          240       2400   172                                            2290          294       600    116                                            #7            250       87     332                                    1000 bp #10           166       53     348                                            #11           264       16     279                                            #4            243       36     277                                     500 bp #5            208       144    266                                            #8            240       1342   215                                    ______________________________________                                    

The results set forth above indicate that bacteria (and by inferenceother hosts) can be protected against infection with an RNA virus byinserting a gene controlling production of an inoperative fragment of aviral RNA replicase enzyme into a host organism.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the inventionas set forth herein.

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What is claimed as new and desired to be secured by Letters Patent ofthe United States is:
 1. A method of making bacterial cells resistant toinfection by a virus, comprising:a) isolating DNA coding for a gene, orfragment thereof, of said virus; b) operably linking said DNA, or afragment thereof, within an expression vector; c) transforming saidbacterial cells with said expression vector; d) culturing saidtransformed bacterial cells in the presence of said virus, wherein saidDNA, or fragment thereof, is expressed as a gene product, and whereinsaid gene product disrupts an essential activity of said virus; and e)identifying said transformed bacterial cells which are resistant toinfection by said virus by selecting said transformed bacterial cellswhich survive or resist infection by said virus.
 2. The method of claim1, wherein said transformed bacterial cells comprise plasmids containingsaid DNA, or fragment thereof, wherein said plasmids are capable ofreplication in said transformed bacterial cells.
 3. The method of claim1, wherein said DNA, or fragment thereof, is inserted into thechromosomes of said bacterial cells.
 4. The method of claim 1, whereinsaid bacterial cells are selected from the group consisting of bacterialcells of the genera Escherichia, Streptococcus, Streptococens andLactobacillus.
 5. The method of claim 4, wherein said bacterial cellsare selected from the group consisting of Escherichia coli,Streptococcus lactis and Streptococens cremoris.
 6. The method of claim1, wherein said DNA is cDNA.
 7. The method of claim 1, wherein saidgene, or fragment thereof, is selected from the group consisting of areplicase gene or fragment thereof, a coat protein gene or fragmentthereof, and a maturation protein gene or fragment thereof.
 8. Themethod of claim 7, wherein said gene or fragment thereof is a replicasegene fragment.
 9. The method of claim 8, wherein said gene or fragmentthereof contains a binding site for a replicase enzyme.
 10. The methodof claim 7, wherein said gene or fragment thereof is a coat protein,maturation protein, or a fragment thereof.
 11. A method of makingcultured plant cells or cultured plant tissue resistant to infection byan RNA virus, comprising:a) isolating DNA coding for a gene, or fragmentthereof, of said virus; b) operably linking said DNA, or a fragmentthereof, within an expression vector; c) transforming said plant cellsor plant tissue with said expression vector; d) growing said transformedplant cells or plant tissue in the presence of said virus, wherein saidDNA, or fragment thereof, is expressed as a gene product, and whereinsaid gene product disrupts an essential activity of said virus; and e)identifying said transformed plant cells or plant tissue which areresistant to infection by said virus by selecting said transformed plantcells or plant tissue which survive or resist infection by said virus.12. The method of claim 11, wherein said DNA or fragment thereof isexpressed in the sense direction.
 13. The method of claim 11, whereinsaid DNA or fragment thereof is expressed in the antisense direction.14. The method of claim 11, wherein said gene or fragment thereof is areplicase gene or fragment thereof.
 15. The method of claim 14, whereinsaid gene or fragment thereof contains a binding site for a replicaseenzyme.
 16. The method of claim 11, wherein said plant cells or planttissue are dicotyledonous.
 17. The method of claim 11, wherein saidexpression vector is an Agrobacterium tumefaciens plasmid.
 18. Themethod of claim 11, wherein said virus is selected from the groupconsisting of alfalfa mosaic virus, brome mosaic virus, barley yellowdwarf virus, beet yellows virus, cucumber mosaic virus, lettuce necroticyellow virus, maize chlorotic dwarf virus, pea enation virus, potatovirus S, potato virus X, potato virus Y, southern bean mosaic virus,tomato ringspot virus, tobacco ringspot virus, tobacco mosaic virus,tobacco streak virus, turnip yellow mosaic virus and wound tumor virus.19. The method of claim 18, wherein said virus is selected from thegroup consisting of tobacco mosaic virus, cucumber mosaic virus, alfalfamosaic virus and tobacco ring spot virus.
 20. The method of claim 11,wherein said plant cells or plant tissue are tomato cells or tissue. 21.The method of claim 11, wherein said plant cells or plant tissue arecotton cells or tissue.
 22. The method of claim 18, wherein said plantcells or plant tissue are potato cells or tissue and said virus ispotato virus X.
 23. The method of claim 18, wherein said plant cells orplant tissue are potato cells or tissue and said virus is potato virusY.
 24. The method of claim 11, wherein said gene or fragment thereof isa coat protein gene or a fragment thereof.
 25. The method of claim 16,wherein said expression vector is an Agrobacterium tumefaciens plasmid.26. The method of claim 25, wherein said virus is selected from thegroup consisting of alfalfa mosaic virus, brome mosaic virus, barleyyellow dwarf virus, beet yellows virus, cucumber mosaic virus, lettucenecrotic yellow virus, maize chlorotic dwarf virus, pea enation Virus,potato virus S, potato virus X, potato virus Y, southern bean mosaicvirus, tomato ring spot virus, tobacco ring spot virus, tobacco mosaicvirus, tobacco streak virus, turnip yellow mosaic virus and wound tumorvirus.
 27. The method of claim 26, wherein said virus is selected fromthe group consisting of tobacco mosaic virus, cucumber mosaic virus,alfalfa mosaic virus and tobacco ring spot virus.
 28. The method ofclaim 16, wherein said plant cells or plant tissue are tomato, cotton orpotato cells or tissue.